Search

Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neurons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the molecular and behavioral consequences of challenging Grhl3PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in trigeminal ganglia (TG), before and after HDM. We found that neither Grhl3PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3PAR2/+ mice. Despite the absence of scratching in untreated Grhl3PAR2/+ mice, several TG genes in these mice were up-regulated compared to WT. HDM treatment of the Grhl3PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was up-regulated in HDM-treated Grhl3PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcriptional changes in IL31Tg mice, demonstrating that a common genetic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.

Abstract Migraine is a major health burden worldwide with complex pathophysiology and multifarious underlying mechanisms. One poorly understood issue concerns the early steps in the generation of migraine pain. To elucidate the basic process of migraine pain further, it seems useful to consider key molecular players that may operate synergistically to evoke headache. While the neuropeptide CGRP is an important contributor, we propose that extracellular ATP (that generally plays a powerful nociceptive role) is also a major component of migraine headache, acting in concert with CGRP to stimulate trigeminal nociceptive neurons. The aim of the present focused review is to highlight the role of ATP activating its P2X3 membrane receptors selectively expressed by sensory neurons including their nerve fiber terminals in the meninges. Specifically, we present data on the homeostasis of ATP and related purines in the trigeminovascular system and in the CNS; the basic properties of ATP signalling at peripheral and central nerve terminals; the characteristics of P2X3 and related receptors in trigeminal neurons; the critical speed and persistence of P2X3 receptor activity; their cohabitation at the so-called meningeal neuro-immune synapse; the identity of certain endogenous agents cooperating with ATP to induce neuronal sensitization in the trigeminal sensory system; the role of P2X3 receptors in familial type migraine; the current state of P2X3 receptor antagonists and their pharmacological perspectives in migraine. It is proposed that the unique kinetic properties of P2X3 receptors activated by ATP offer an interesting translational value to stimulate future studies for innovative treatments of migraine pain.

Migraine is a major health burden worldwide with complex pathophysiology and multifarious underlying mechanisms. One poorly understood issue concerns the early steps in the generation of migraine pain. To elucidate the basic process of migraine pain further, it seems useful to consider key molecular players that may operate synergistically to evoke headache. While the neuropeptide CGRP is an important contributor, we propose that extracellular ATP (that generally plays a powerful nociceptive role) is also a major component of migraine headache, acting in concert with CGRP to stimulate trigeminal nociceptive neurons. The aim of the present focused review is to highlight the role of ATP activating its P2X3 membrane receptors selectively expressed by sensory neurons including their nerve fiber terminals in the meninges. Specifically, we present data on the homeostasis of ATP and related purines in the trigeminovascular system and in the CNS; the basic properties of ATP signalling at peripheral and central nerve terminals; the characteristics of P2X3 and related receptors in trigeminal neurons; the critical speed and persistence of P2X3 receptor activity; their cohabitation at the so-called meningeal neuro-immune synapse; the identity of certain endogenous agents cooperating with ATP to induce neuronal sensitization in the trigeminal sensory system; the role of P2X3 receptors in familial type migraine; the current state of P2X3 receptor antagonists and their pharmacological perspectives in migraine. It is proposed that the unique kinetic properties of P2X3 receptors activated by ATP offer an interesting translational value to stimulate future studies for innovative treatments of migraine pain. [ABSTRACT FROM AUTHOR]

Trigeminal ganglion (TG) is the first station of sensory pathways in the orofacial region. The TG neurons communicate with satellite glial cells (SGCs), macrophages and other cells forming a functional unit that is responsible for processing of orofacial sensory information. Purinergic signaling, one of the most widespread autocrine and paracrine pathways, plays a crucial role in intercellular communication. The multidirectional action of purinergic signaling in different cell types contributes to the neuromodulation and orofacial sensation. To fully understand the purinergic signaling in these processes, it is essential to determine the shared and unique expression patterns of genes associated with purinergic signaling in different cell types. Here, we performed single-cell RNA sequencing of 22,969 cells isolated from normal mouse TGs. We identified 18 distinct cell populations, including 6 neuron subpopulations, 3 glial subpopulations, 7 immune cell subpopulations, fibroblasts, and endothelial cells. We also revealed the transcriptional features of genes associated with purinergic signaling, including purinergic receptors, extracellular adenosine triphosphate (eATP) release channels, eATP metabolism-associated enzymes, and eATP transporters in each cell type. Our results have important implications for understanding and predicting the cell type-specific roles of the purinergic signaling in orofacial signal processing in the trigeminal primary sensory system. [ABSTRACT FROM AUTHOR]

IB4-positive maxillary trigeminal ganglion (TG) neurons are a subtype of afferent neurons involving nociception in orofacial regions, and excitability of these neurons is associated with orofacial nociceptive sensitivity. TREK-2 channel is a member of two-pore domain potassium (K2P) channel family mediating leak K+ currents. It has been shown previously that TREK-2 channel activity can be enhanced following GABAB receptor activation, leading to a reduction of cortical neuron excitability. In the present study, we have characterized TREK-2 channel expression on maxillary TG neurons and investigated the effect of the GABAB agonist baclofen on electrophysiological properties of small-sized maxillary TG neurons of rats. We show with immunohistochemistry that TREK-2 channels are predominantly expressed in small-sized IB4-positive maxillary TG neurons. Patch-clamp recordings on neurons in ex vivo TG preparations show that baclofen hyperpolarizes resting membrane potentials, increases outward leak currents, and decreases input resistances in IB4-positive maxillary TG neurons. Moreover, baclofen significantly reduces action potential (AP) firing in IB4-positive maxillary TG neurons. In contrast, baclofen shows no significant effect on electrophysiological properties of small-sized nociceptive-like and non-nociceptive-like maxillary trigeminal neurons that are IB4-negatve. Our results suggest that TREK-2 channel activity can be enhanced by baclofen, leading to reduced excitability of IB4-positive maxillary TG neurons. This finding provides new insights into the role of TREK-2 and GABAB receptors in controlling nociceptive sensitivity in orofacial regions, which may have therapeutic implications. [ABSTRACT FROM AUTHOR]

• Benzalkonium chloride induces toxicity on human corneal epithelial cells. • Conditioned medium from corneal cells exposed to BAK (BAK-CM) triggers neuronal genes. • BAK-CM enhances neuronal damage and inflammatory genes at an early stage. • BAK-CM activates pro-survival pathways in neurons at a late stage. Benzalkonium chloride (BAK), a quaternary ammonium compound widely used as disinfecting agent as well as preservative in eye drops is known to induce toxic effects on the ocular surface with inflammation and corneal nerve damage leading to dry eye disease (DED) in the medium-to-long term. The aim of this study was to evaluate in vitro the toxicity of a conditioned medium produced by corneal epithelial cells previously exposed to BAK (BAK-CM) on trigeminal neuronal cells. A human corneal epithelial (HCE) cell line was exposed to 5.10−3% BAK (i.e. 0.005% BAK) for 15 min and let recover for 5 h to prepare a BAK-CM. This BAK concentration is the lowest one found in eye drops. After this recovery period, BAK effect on HCE cells displayed cytotoxicity, morphological alteration, apoptosis, oxidative stress, ATP release, CCL2 and IL6 gene induction, as well as an increase in CCL2, IL-6 and MIF release. Next, a mouse trigeminal ganglion primary culture was exposed to the BAK-CM for 2 h, 4 h or 24 h. Whereas BAK-CM did not alter neuronal cell morphology, or induced neuronal cytotoxicity or oxidative stress, BAK-CM induced gene expression of Fos (neuronal activation marker), Atf3 (neuronal injury marker), Ccl2 and Il6 (inflammatory markers). Two and 4 h BAK-CM exposure promoted a neuronal damage (ATF-3, phospho-p38 increases; phospho-Stat3 decreases) while 24 h-BAK-CM exposure initiated a prosurvival pathway activation (phospho-p44/42, phospho-Akt increases; ATF-3, GADD153, active Caspase-3 decreases). In conclusion, this in vitro model, simulating paracrine mechanisms, represents an interesting tool to highlight the indirect toxic effects of BAK or any other xenobiotic on corneal trigeminal neurons and may help to better understand the cellular mechanisms that occur during DED pathophysiology. [ABSTRACT FROM AUTHOR]

In mice, spared nerve injury replicates symptoms of human neuropathic pain and induces upregulation of many genes in somatosensory neurons. Here we used single cell transcriptomics to probe the effects of partial infraorbital transection of the trigeminal nerve at the cellular level. Uninjured neurons were unaffected by transection of major nerve branches, segregating into many different classes. In marked contrast, axotomy rapidly transformed damaged neurons into just two new and closely-related classes where almost all original identity was lost. Remarkably, sensory neurons also adopted this transcriptomic state following various minor peripheral injuries. By genetically marking injured neurons, we showed that the injury-induced transformation was reversible, with damaged cells slowly reacquiring normal gene expression profiles. Thus, our data expose transcriptomic plasticity, previously thought of as a driver of chronic pain, as a programed response to many types of injury and a potential mechanism for regulating sensation during wound healing.

Introduction: The trigeminal ganglion is unique among the somatosensory ganglia regarding its topography, structure, composition and possibly some functional properties of its cellular components. Being mainly responsible for the sensory innervation of the anterior regions of the head, it is a major target for headache research. One intriguing question is if the trigeminal ganglion is merely a transition site for sensory information from the periphery to the central nervous system, or if intracellular modulatory mechanisms and intercellular signaling are capable of controlling sensory information relevant for the pathophysiology of headaches.Methods: An online search based on PubMed was made using the keyword "trigeminal ganglion" in combination with "anatomy", "headache", "migraine", "neuropeptides", "receptors" and "signaling". From the relevant literature, further references were selected in view of their relevance for headache mechanisms. The essential information was organized based on location and cell types of the trigeminal ganglion, neuropeptides, receptors for signaling molecules, signaling mechanisms, and their possible relevance for headache generation.Results: The trigeminal ganglion consists of clusters of sensory neurons and their peripheral and central axon processes, which are arranged according to the three trigeminal partitions V1-V3. The neurons are surrounded by satellite glial cells, the axons by Schwann cells. In addition, macrophage-like cells can be found in the trigeminal ganglion. Neurons express various neuropeptides, among which calcitonin gene-related peptide is the most prominent in terms of its prevalence and its role in primary headaches. The classical calcitonin gene-related peptide receptors are expressed in non-calcitonin gene-related peptide neurons and satellite glial cells, although the possibility of a second calcitonin gene-related peptide receptor in calcitonin gene-related peptide neurons remains to be investigated. A variety of other signal molecules like adenosine triphosphate, nitric oxide, cytokines, and neurotrophic factors are released from trigeminal ganglion cells and may act at receptors on adjacent neurons or satellite glial cells.Conclusions: The trigeminal ganglion may act as an integrative organ. The morphological and functional arrangement of trigeminal ganglion cells suggests that intercellular and possibly also autocrine signaling mechanisms interact with intracellular mechanisms, including gene expression, to modulate sensory information. Receptors and neurotrophic factors delivered to the periphery or the trigeminal brainstem can contribute to peripheral and central sensitization, as in the case of primary headaches. The trigeminal ganglion as a target of drug action outside the blood-brain barrier should therefore be taken into account. [ABSTRACT FROM AUTHOR]

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross‐communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca2+ influx in trigeminal neurons and the involvement of the different types of voltage‐gated Ca2+ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca2+ influx, and a slower component triggered by Ca2+ influx. The Ca2+‐dependent component was inhibited 80% by ω‐conotoxin‐MVIIC, an inhibitor of both N‐ and P/Q‐type Cav channels, and 55% by the P/Q‐type selective inhibitor ω‐agatoxin‐IVA. The selective L‐type Ca2+ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N‐ and P/Q‐, but not L‐type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N‐ and P/Q‐type channels may exert their function by also modulating the somatic release and cross‐communication between sensory neurons. In this study we addressed the dependence of somatic release on Ca2+ influx in trigeminal neurons, and the involvement of the different types of voltage‐gated Ca2+ (Cav) channels in the process. We found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca2+ influx, and a slower component triggered by Ca2+ influx though N‐ and P/Q‐type Cav channels. [ABSTRACT FROM AUTHOR]

Ameloblastomas are locally invasive and aggressive odontogenic tumors treated via surgical resection, which results in facial deformity and significant morbidity. Few studies have addressed the cellular and molecular events of ameloblastoma onset and progression, thus hampering the development of non-invasive therapeutic approaches. Tumorigenesis is driven by a plethora of factors, among which innervation has been long neglected. Recent findings have shown that innervation directly promotes tumor progression. On this basis, we investigated the molecular characteristics and neurotrophic properties of human ameloblastomas. Our results showed that ameloblastomas express dental epithelial stem cell markers, as well as components of the Notch signaling pathway, indicating persistence of stemness. We demonstrated that ameloblastomas express classical stem cell markers, exhibit stem cell potential, and form spheres. These tumors express also molecules of the Notch signaling pathway, fundamental for stem cells and their fate. Additionally, we showed that ameloblastomas express the neurotrophic factors NGF and BDNF, as well as their receptors TRKA, TRKB, and P75/NGFR, which are responsible for their innervation by trigeminal axons in vivo. In vitro studies using microfluidic devices showed that ameloblastoma cells attract and form connections with these nerves. Innervation of ameloblastomas might play a key role in the onset of this malignancy and might represent a promising target for non-invasive pharmacological interventions.

Hydrogen sulfide (H2S), a well-established member of the gasotransmitter family, is involved in a variety of physiological functions, including pro-nociceptive action in the sensory system. Although several reports have shown that H2S activates sensory neurons, the molecular targets of H2S action in trigeminal (TG) nociception, implicated in migraine, remains controversial. In this study, using suction electrode recordings, we investigate the effect of the H2S donor, sodium hydrosulfide (NaHS), on nociceptive firing in rat meningeal TG nerve fibers. The effect of NaHS was also explored with patch-clamp and calcium imaging techniques on isolated TG neurons. NaHS dramatically increased the nociceptive firing in TG nerve fibers. This effect was abolished by the TRPV1 inhibitor capsazepine but was partially prevented by the TRPA1 blocker HC 030031. In a fraction of isolated TG neurons, NaHS transiently increased amplitude of capsaicin-induced currents. Moreover, NaHS by itself induced inward currents in sensory neurons, which were abolished by the TRPV1 inhibitor capsazepine suggesting involvement of TRPV1 receptors. In contrast, the inhibitor of TRPA1 receptors HC 030031 did not prevent the NaHS-induced currents. Imaging of a large population of TG neurons revealed that NaHS induced calcium transients in 41% of tested neurons. Interestingly, this effect of NaHS in some neurons was inhibited by the TRPV1 antagonist capsazepine whereas in others it was sensitive to the TRPA1 blocker HC 030031. Our data suggest that both TRPV1 and TRPA1 receptors play a role in the pro-nociceptive action of NaHS in peripheral TG nerve endings in meninges and in somas of TG neurons. We propose that activation of TRPV1 and TRPA1 receptors by H2S during neuro-inflammation conditions contributes to the nociceptive firing in primary afferents underlying migraine pain.

Hydrogen sulfide (H2S), a well-established member of the gasotransmitter family, is involved in a variety of physiological functions, including pro-nociceptive action in the sensory system. Although several reports have shown that H2S activates sensory neurons, the molecular targets of H2S action in trigeminal (TG) nociception, implicated in migraine, remains controversial. In this study, using suction electrode recordings, we investigate the effect of the H2S donor, sodium hydrosulfide (NaHS), on nociceptive firing in rat meningeal TG nerve fibers. The effect of NaHS was also explored with patch-clamp and calcium imaging techniques on isolated TG neurons. NaHS dramatically increased the nociceptive firing in TG nerve fibers. This effect was abolished by the TRPV1 inhibitor capsazepine but was partially prevented by the TRPA1 blocker HC 030031. In a fraction of isolated TG neurons, NaHS transiently increased amplitude of capsaicin-induced currents. Moreover, NaHS by itself induced inward currents in sensory neurons, which were abolished by the TRPV1 inhibitor capsazepine suggesting involvement of TRPV1 receptors. In contrast, the inhibitor of TRPA1 receptors HC 030031 did not prevent the NaHS-induced currents. Imaging of a large population of TG neurons revealed that NaHS induced calcium transients in 41% of tested neurons. Interestingly, this effect of NaHS in some neurons was inhibited by the TRPV1 antagonist capsazepine whereas in others it was sensitive to the TRPA1 blocker HC 030031. Our data suggest that both TRPV1 and TRPA1 receptors play a role in the pro-nociceptive action of NaHS in peripheral TG nerve endings in meninges and in somas of TG neurons. We propose that activation of TRPV1 and TRPA1 receptors by H2S during neuro-inflammation conditions contributes to the nociceptive firing in primary afferents underlying migraine pain. [ABSTRACT FROM AUTHOR]

Black peppercorns (Piper nigrum L.) elicit a pungent and tingling oral impression. Their pungency is partially explained by the agonist activity of some of their active principles, especially piperine, on TRP channels. However, we recently showed that piperine, as well as other pungent compounds, also possess a marked effect on two-pore domain (KCNK, K2P) K+ channels. Members of this family play a key role in maintaining the resting membrane potential of excitable cells. Interestingly, tingling compounds have been shown to induce neuronal excitation by inhibiting KCNK channels. We addressed the question of whether it was plausible that KCNK channels could constitute a physiologically relevant target for the sensory active compounds present in black peppercorns. Because previous studies have demonstrated that mouse trigeminal neurons respond to several pungent compounds, to which humans are also sensitive, we used a primary culture of mouse trigeminal neurons to investigate whether the effect of piperine on these cell types could also be mediated by KCNK channels. We observed that even in the presence of classical TRP-antagonists, piperine was still able to activate a fraction of trigeminal neurons. Furthermore, our results showed that piperine is capable of inducing neuronal depolarization by a mechanism that does not require extracellular Na+ or Ca2+. This depolarization was mediated by the inhibition of a background K+ conductance, most likely corresponding to the KCNK channels of the TASK subfamily. We then performed a screening with 12 other pungent and/or tingling chemosensates isolated from black peppercorns. These compounds were evaluated on Xenopus laevis oocytes expressing the human orthologues of KCNK3, KNCK9 and KCNK18, which we previously showed to be inhibited by piperine. Remarkably, almost all of the isolated chemosensates inhibited the basal activity of hKCNK3, with 1-(octadeca-2E,4E,13/12Ztrienoyl) pyrrolidine acting as one of the most potent natural blockers for hKCNK3 found to date. Our results suggest that KCNK channels, especially KCNK3, are likely to play a complementary role to TRP channels in the complex orosensory impression elicited by black peppercorns, while they also help to expand the pharmacological knowledge of KCNK channels. [ABSTRACT FROM AUTHOR]

The monoamine neurotransmitter serotonin (5-HT) and the neuropeptide calcitonin gene-related peptide (CGRP) play an important role in migraine pathophysiology. To study potential interplay between 5-HT and CGRP in peripheral trigeminal nociception, we performed calcium imagining and patch clamp studies in rat trigeminal ganglia cells. We found that 5-HT activated Ca transients in 18 % of trigeminal ganglia neurons. Exposure of trigeminal cells to CGRP significantly increased the number of 5-HT positive cells to 35 % and increased the amplitude of 5-HT-induced Ca transients. Using patch clamp technique, we show that 37 % percent of trigeminal cells generated desensitizing membrane currents suggesting functional expression of 5-HT3 receptors. These responses were partially co-localized either with ATP-gated or capsaicin-sensitive neurons. Exposure to CGRP for 2 h increased the current density in the ATP-sensitive fraction of trigeminal neurons. Taken together, these data suggest that 5-HT receptor sensitization contributes to the pro-nociceptive effect of CGRP in trigeminal neurons. [ABSTRACT FROM AUTHOR]

Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pro-nociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pro-nociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain. [ABSTRACT FROM AUTHOR]

Peripheral nerves innervating the mucosae of the nose, mouth, and throat protect the organism against chemical hazards. Upon their stimulation, characteristic perceptions (e.g., stinging and burning) and various reflexes are triggered (e.g., sneezing and cough). The potency of a chemical to cause sensory irritation can be estimated by a mouse bioassay assessing the concentration-dependent decrease in the respiratory rate (50 % decrease: RD). The involvement of the N. trigeminus and its sensory neurons in the irritant-induced decrease in respiratory rates are not well understood to date. In calcium imaging experiments, we tested which of eight different irritants (RD 5-730 ppm) could induce responses in primary mouse trigeminal ganglion neurons. The tested irritants acetophenone, 2-ethylhexanol, hexyl isocyanate, isophorone, and trimethylcyclohexanol stimulated responses in trigeminal neurons. Most of these responses depended on functional TRPA1 or TRPV1 channels. For crotyl alcohol, 3-methyl-1-butanol, and sodium metabisulfite, no activation could be observed. 2-ethylhexanol can activate both TRPA1 and TRPV1, and at low contractions (100 µM) G protein-coupled receptors (GPCRs) seem to be involved. GPCRs might also be involved in the mediation of the responses to trimethylcyclohexanol. By using neurobiological tools, we showed that sensory irritation in vivo could be based on the direct activation of TRP channels but also on yet unknown interactions with GPCRs present in trigeminal neurons. Our results showed that the potency suggested by the RD values was not reflected by direct nerve-compound interaction. [ABSTRACT FROM AUTHOR]

Impaired corneal innervation and sensitivity are the main causes of corneal neurotrophic keratopathy which simultaneously also leads to poor epithelial wound healing. Restoration of the diminished communication between the corneal epithelium and trigeminal nerve is indispensable for the proper functioning of the epithelium. The present study aims to investigate corneal epithelial and trigeminal neuron interactions to shed light on corneal wound healing during neurotrophic keratopathy. Mouse trigeminal neurons and corneal epithelial cells were cultured according to standard methods. To study the effect of corneal epithelial cells on trigeminal neurons as well as the effect of trigeminal neurons on corneal epithelial cells during wound healing, conditioned media from the cultures of pure trigeminal neurons (CNM) and corneal epithelial cells (CEM) were collected freshly and applied on the other cell type. Neurite outgrowth assay and RT-PCR analysis using primers specific for substance P (SP), Map1a, Map1b were performed on trigeminal neurons in the presence of CEM. We observed an increase in the neurite outgrowth in the presence of CEM and also in co-culture with corneal epithelial cells. Increase in the expression of SP mRNA and a decrease in the expression of Map1b mRNA was observed in the presence of CEM. We also observed the presence of epithelial-to-mesenchymal transition (EMT)-like phenomenon during wound healing using a scratch assay in primary corneal epithelial cultures. This system was further employed to study the effect of CNM on corneal epithelial cells in the context of wound healing to find the effect of trigeminal neurons on epithelial cells. RT-PCR analysis of Pax6 expression in corneal epithelial cell cultures with scratch served as a positive control. Further, we also show the expression of bone morphogenetic protein 7 (BMP7) mRNA in corneal epithelial cells which is decreased gradually along with Pax6 mRNA when cultured together in the presence of CNM. The expression and down regulation of BMP7 in the presence of CNM was further confirmed at the protein level by western blotting. From this study it seems that the epithelial and neuronal interactions in the cornea may contribute to the corneal innervation as well as recovery of corneal epithelial cells during injury. Appraising the differences in the expression of various signalling molecules during EMT of epithelial cells in the presence of SP and BMP7 gives an insight into the detailed dissection of the involved signalling pathways to develop future therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Endodontic sealers are selected on the basis of their antimicrobial properties and ability to provide a tight seal. Sealer extrusions, whether intentional or unintentional, are common during obturation procedures. Such events have been correlated with increased postoperative discomfort and persistent pain states. However, the mechanisms underlying this phenomenon are largely unknown. Thus, we sought to evaluate the effect of commonly used endodontic sealers on peripheral nociceptors. We hypothesized that endodontic sealers can directly activate trigeminal nociceptors in a concentration-dependent manner, resulting in release of calcitonin gene-related peptide (CGRP), a potent modulator of neurogenic inflammation. Methods: Rat trigeminal sensory neurons were exposed in vitro to vehicle, zinc oxide-eugenol (ZOE)–based sealer, AH Plus, EndoSequence BC sealer, or RealSeal SE. Neuronal activation was measured by quantification of neuropeptide (CGRP) release. In addition, cultured neurons were also subjected to the set form of all 4 sealers. The concentration of CGRP released was quantified by using a radioimmunoassay. Data were analyzed by using one-way analysis of variance with Newman-Keuls multiple comparison post hoc test. Results: Both ZOE-based sealer and AH Plus in their fresh form evoked greater CGRP release than the control groups. Conversely, EndoSequence BC and RealSeal sealers both reduced basal GCRP release at all concentrations tested. Evaluation of the set sealers revealed that only ZOE-based sealer evoked significant CGRP release compared with its control group. Conclusions: Overall, our results suggest that sealers can directly activate trigeminal nociceptors, leading to a robust release of CGRP, and may therefore lead to pain and neurogenic inflammation. This direct activation along with the immunologic response may underlie the symptoms and flare-up occurrences often seen with sealer extrusions. [Copyright &y& Elsevier]

Trigeminal ganglion (TG) is the first station of sensory pathways in the orofacial region. The TG neurons communicate with satellite glial cells (SGCs), macrophages and other cells forming a functional unit that is responsible for processing of orofacial sensory information. Purinergic signaling, one of the most widespread autocrine and paracrine pathways, plays a crucial role in intercellular communication. The multidirectional action of purinergic signaling in different cell types contributes to the neuromodulation and orofacial sensation. To fully understand the purinergic signaling in these processes, it is essential to determine the shared and unique expression patterns of genes associated with purinergic signaling in different cell types. Here, we performed single-cell RNA sequencing of 22,969 cells isolated from normal mouse TGs. We identified 18 distinct cell populations, including 6 neuron subpopulations, 3 glial subpopulations, 7 immune cell subpopulations, fibroblasts, and endothelial cells. We also revealed the transcriptional features of genes associated with purinergic signaling, including purinergic receptors, extracellular adenosine triphosphate (eATP) release channels, eATP metabolism-associated enzymes, and eATP transporters in each cell type. Our results have important implications for understanding and predicting the cell type-specific roles of the purinergic signaling in orofacial signal processing in the trigeminal primary sensory system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jia, Liu, Chu, Liu, Mai and Fan.)

Highlights: [•] CSD enhanced lipid peroxidation in cortex, meninges and trigeminal ganglia. [•] H2O2 enhanced spiking of trigeminal nerves and promoted CGRP release. [•] Effect of H2O2 on spiking was abolished by the TRPA1 antagonist. [•] Oxidative stress could couple CSD with activation of the trigeminovascular system. [ABSTRACT FROM AUTHOR]

Abstract: Enhanced nociceptive firing in trigeminal ganglion neurons is a likely reason for migraine pain. In experimental migraine-like conditions induced by the calcitonin gene-related peptide (CGRP), P2X3 receptors abundantly expressed in trigeminal neurons are highly responsive to the excitatory action of extracellular ATP. In this study, we tested whether naproxen, a common antimigraine medicine, could affect the function of P2X3 receptors in the presence or absence of the algogen nerve growth factor (NGF), the level of which is elevated in patients with chronic migraine. We used calcium imaging and patch clamp recordings from rat trigeminal neurons, which were activated by a relative specific P2X3 agonist α,β-meATP or by high potassium-induced depolarization. In the absence of NGF, naproxen dose-dependently (0.1–1 mM) reduced intracellular calcium transients elicited by α,β-meATP. Naproxen also led to a slight, but significant, reduction in calcium transients induced by potassium ions, indicating the involvement of voltage-gated calcium channels. The inhibitory action of 1 mM naproxen was enhanced after NGF pretreatment, suggesting that P2X3 receptors in sensitized neurons are more susceptible to inhibition by high doses of this nonsteroidal anti-inflammatory drug (NSAID). Using patch clamp recordings from HEK293 cells expressing P2X3 receptors, we tested the direct action of naproxen on P2X3 receptor-mediated membrane currents. In clinically relevant concentrations of 0.5 mM, naproxen produced a use-dependent blocking effect on ATP receptors. Kinetic analysis suggests that naproxen inhibited P2X3 receptors via facilitation of fast desensitization, which determines current decay in the continuous presence of the agonist. In summary, we present a novel fast mechanism for the antimigraine action of naproxen, which can act in synergy with the cyclooxygenase inhibition to attenuate headaches. [Copyright &y& Elsevier]

Er,Cr:YSGG lasers are used clinically in dentistry. The advantages of laser therapy include minimal thermal damage and the alleviation of pain. This study examined whether the Er,Cr:YSGG laser has in vivo and in vitro antinociceptive effects in itself. In capsaicin-evoked acute licking/shaking tests and Hargreaves tests, laser irradiation with an aerated water spray suppressed nociceptive behavior in mice. Laser irradiation attenuated TRPV1 activation by capsaicin in Ca2+ imaging experiments with TRPV1-overexpressing cells and cultured trigeminal neurons. Therefore, the laser-induced behavioral changes are probably due to the loss of TRPV1 activity. TRPV4 activity was also attenuated, but limited mechanical antinociception by the laser was observed. The laser failed to alter the other receptor functions, which indicates that the antinociceptive effect of the laser is dependent on TRPV1. These results suggest that the Er,Cr:YSGG laser has analgesic effects via TRPV1 inhibition. Such mechanistic approaches may help define the laser-sensitive pain modality and increase its beneficial uses. [ABSTRACT FROM PUBLISHER]

Abstract: Trigeminal nerve fibers in nasal and oral cavities are sensitive to various environmental hazardous stimuli, which trigger many neurotoxic problems such as chronic migraine headache and trigeminal irritated disorders. However, the role of JNK kinase cascade and its epigenetic modulation of histone remodeling in trigeminal ganglion (TG) neurons activated by environmental neurotoxins remains unknown. Here we investigated the role of JNK/c-Jun cascade in the regulation of acetylation of H3 histone in TG neurons following in vitro stimulation by a neuro-inflammatory agent, mustard oil (MO). We found that MO stimulation elicited JNK/c-Jun pathway significantly by enhancing phospho-JNK1, phospho-c-Jun expression, and c-Jun activity, which were correlated with an elevated acetylated H3 histone in TG neurons. However, increases in phospho-c-Jun and c-Jun activity were significantly blocked by a JNK inhibitor, SP600125. We also found that altered H3 histone remodeling, assessed by H3 acetylation in triggered TG neurons, was reduced by SP600125. The study suggests that the activated JNK signaling in regulation of histone remodeling may contribute to neuro-epigentic changes in peripheral sensory neurons following environmental neurotoxic exposure. [Copyright &y& Elsevier]

Migraine headache originates from the stimulation of nerve terminals of trigeminal ganglion neurons that innervate meninges. Characteristic features of migraine pain are not only its delayed onset but also its persistent duration. Current theories propose that endogenous substances released during a migraine attack (the neuropeptide calcitonin gene-related peptide [CGRP] and the neurotrophin nerve growth factor [NGF]) sensitize trigeminal neurons to transmit nociceptive signals to the brainstem, though the mechanisms remain poorly understood. Recent studies indicate that acute, long-lasting sensitization of trigeminal nociceptive neurons occurs via distinct processes involving enhanced expression and function of adenosine triphosphate (ATP)-gated P2X3 receptors known to play a role in chronic pain. In particular, on cultured trigeminal neurons, CGRP (via protein kinase A-dependent signaling) induces a slowly developing upregulation of the ionic currents mediated by P2X3 receptors by enhancing receptor trafficking to the neuronal membrane and activating their gene transcription. Such upregulated receptors acquire the ability to respond repeatedly to extracellular ATP, thus enabling long-lasting signaling of painful stimuli. In contrast, NGF induces rapid, reversible upregulation of P2X3 receptor function via protein kinase C phosphorylation, an effect counteracted by anti-NGF antibodies. The diverse intracellular signaling pathways used by CGRP and NGF show that the sensitization of P2X3 receptor function persists if the action of only one of these migraine mediators is blocked. These findings imply that inhibiting a migraine attack might be most efficient by a combinatorial approach. The different time domains of P2X3 receptor modulation by NGF and CGRP suggest that the therapeutic efficacy of novel antimigraine drugs depends on the time of administration. [ABSTRACT FROM AUTHOR]

Six transient receptor potential (TRP) ion channels expressed in the sensory afferents play an important role as body thermosensors and also as peripheral pain detectors. It is known that a number of natural compounds specifically activate those sensory neuronal TRP channels, and a well-known example is cinnamaldehyde for TRPA1. Here we show that human and mouse TRPA1 are activated by acetaldehyde, an intermediate substance of ethanol metabolism, in the HEK293T cell heterologous expression system and in cultured mouse trigeminal neurons. Acetaldehyde failed to activate other temperature-sensitive TRP channels expressed in sensory neurons. TRPA1 antagonists camphor and gadolinium, and a general TRP blocker ruthenium red inhibited TRPA1 activation by acetaldehyde. Camphor, gadolinium and ruthenium red also suppressed the acute nociceptive behaviors induced by the intradermal administration of acetaldehyde into the mouse footpads. Intradermal co-application of prostaglandin E2 and acetaldehyde greatly potentiated the acetaldehyde-induced nociceptive responses, and this effect was reversed by treatment with the TRPA1 antagonist camphor. These results suggest that acetaldehyde causes nociception via TRPA1 activation. Our data may also help elucidate the mechanisms underlying acetaldehyde-related pathological symptoms such as hangover pain. [ABSTRACT FROM AUTHOR]

Abstract: Temperature-activated transient receptor potential ion channels (thermoTRPs) are known to function as ambient temperature sensors and are also involved in peripheral pain sensation. The thermoTRPs are activated by a variety of chemicals, of which specific activators have been utilized to explore the physiology of particular channels and sensory nerve subtypes. The use of capsaicin for TRPV1 is an exemplary case for nociceptor studies. In contrast, specific agents for another vanilloid subtype channel, TRPV2 have been lacking. Here, we show that probenecid is able to activate TRPV2 using electrophysiological and calcium imaging techniques with TRPV2-expressing HEK293T cells. Five other sensory thermoTRPs-TRPV1, TRPV3, TRPV4, TRPM8 and TRPA1-failed to show a response to this drug in the same heterologous expression system, suggesting that probenecid is a specific activator for TRPV2. Probenecid-evoked responses were also reproduced in a distinct subset of cultured trigeminal neurons that were responsive to 2-aminoethoxydiphenyl borate, a TRPV1-3 activator. The probenecid-sensitive neurons were mainly distributed in a medium to large-diameter population, in agreement with previous observations with TRPV2 immunolocalization. Under inflammation, probenecid elicited nociceptive behaviors in in vivo assays. These results suggest that TRPV2 is specifically activated by probenecid and that this chemical might be useful for investigation of pain-related TRPV2 function. [Copyright &y& Elsevier]

TRPA1 is a calcium-permeable nonselective cation transient receptor potential (TRP) channel that functions as an excitatory ionotropic receptor in nociceptive neurons. TRPA1 is robustly activated by pungent substances in mustard oil, cinnamon, and garlic and mediates the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate a bimodal sensitivity of TRPA1 to menthol, a widely used cooling agent and known activator of the related cold receptor TRPM8. In whole-cell and single-channel recordings of heterologously expressed TRPA1, submicromolar to low-micromolar concentrations of menthol cause channel activation, whereas higher concentrations lead to a reversible channel block. In addition, we provide evidence for TRPA1-mediated menthol responses in mustard oil-sensitive trigeminal ganglion neurons. Our data indicate that TRPA1 is a highly sensitive menthol receptor that very likely contributes to the diverse psychophysical sensations after topical application of menthol to the skin or mucous membranes of the oral and nasal cavities. [ABSTRACT FROM AUTHOR]

Abstract: Herpes simplex virus type-1 (HSV-1) primarily infects mucoepithelial tissues of the eye and the orofacial region. Subsequently, the virus is retrogradely transported through the axons of the trigeminal sensory neurons to their nuclei, where the virus establishes a life-long latent infection. During this latency period, the viral genome is transcriptionally silent except for a single region encoding the latency-associated transcript (LAT). To understand how HSV-1 latency might affect the expression of substance P in sensory neurons, we transfected primary cultures of trigeminal neurons obtained from rat embryos, with LAT expressing plasmids. The expression of LAT increased the percentage of substance P-immunoreactive neurons by two thirds. To examine the effect of bone morphogenetic protein-7 (BMP7) on the LAT-induced increase in substance P expression in trigeminal neurons, cultures transfected with LAT were treated with BMP7. Treatment with BMP7 reversed the effects of LAT on substance P expression in trigeminal neurons. Our data show for the first time that LAT increases substance P expression in trigeminal neurons and BMP7 can reverse these effects of LAT. [Copyright &y& Elsevier]

Single-channel currents from Na-dependent K channels (K) were recorded from cell-attached and inside-out membrane patches of cultured avian trigeminal ganglion neurons by means of the patchclamp technique. Single-channel properties, such as the high elementary conductance and the occurrence of subconductance levels, were unchanged after the patches had been excised from the cells, indicating that they are not under the control of soluble cytoplasmic factors. In cellattached recordings at the cell resting potential the degree of K activity, measured as the probability of the channel being open, P, was low in most cases (around 0.01) and similar to that observed in the inside-out configuration when the bath solution contained concentrations of Na around 30 mM and of K close to the physiological intracellular levels. However, in some cell-attached patches P was high (around 0.2) and comparable to the values measured in cell-free recordings with high Na concentrations in the bath (100 mM). The excision of a highactivity patch in the presence of 30 mM Na resulted in a fall of P in about 20 s, which is consistent with the wash-out of a soluble cytoplasmic molecule. After the excision all K displayed a similar Na sensitivity, irrespective of the degree of activation observed in the cellattached mode. In inside-out patches the P values observed in the presence of either low or high concentrations of Na in bath solutions were not modified by internal Ca (0.8-8.5 μM). The variable degree of K activation observed in cell-attached recordings suggests that either internal Na concentrations reach very high levels close to the membrane, or soluble factor(s) are involved in the modulation of K activity: under such conditions, the Na-activated K current may contribute to the maintenance of the resting membrane potential and to control neuronal membrane excitability. [ABSTRACT FROM AUTHOR]

IB4-positive maxillary trigeminal ganglion (TG) neurons are a subtype of afferent neurons involving nociception in orofacial regions, and excitability of these neurons is associated with orofacial nociceptive sensitivity. TREK-2 channel is a member of two-pore domain potassium (K2P) channel family mediating leak K + currents. It has been shown previously that TREK-2 channel activity can be enhanced following GABAB receptor activation, leading to a reduction of cortical neuron excitability. In the present study, we have characterized TREK-2 channel expression on maxillary TG neurons and investigated the effect of the GABAB agonist baclofen on electrophysiological properties of small-sized maxillary TG neurons of rats. We show with immunohistochemistry that TREK-2 channels are predominantly expressed in small-sized IB4-positive maxillary TG neurons. Patch-clamp recordings on neurons in ex vivo TG preparations show that baclofen hyperpolarizes resting membrane potentials, increases outward leak currents, and decreases input resistances in IB4-positive maxillary TG neurons. Moreover, baclofen significantly reduces action potential (AP) firing in IB4-positive maxillary TG neurons. In contrast, baclofen shows no significant effect on electrophysiological properties of small-sized nociceptive-like and non-nociceptive-like maxillary trigeminal neurons that are IB4-negatve. Our results suggest that TREK-2 channel activity can be enhanced by baclofen, leading to reduced excitability of IB4-positive maxillary TG neurons. This finding provides new insights into the role of TREK-2 and GABAB receptors in controlling nociceptive sensitivity in orofacial regions, which may have therapeutic implications.

Biomedical prosthetics utilizing electrical stimulation have limited, effective spatial resolution due to spread of electrical currents to surrounding tissue, causing nonselective stimulation. So, precise spatial resolution is not possible for traditional neural prosthetic devices, such as cochlear implants. More recently, alternative methods utilize optical stimulation, mainly infrared, sometimes paired with nanotechnology for stimulating action potentials. Infrared stimulation has its own drawbacks, as it may cause collateral heating of surrounding tissue. In previous work, we employed a plasmonic method for stimulation of an electrically excitable neuroblastoma cell line, which had limited success. Here, we report the development of a hybrid electro-plasmonic stimulation platform for spatially and temporally precise neural excitation to address the above deficiencies. Primary trigeminal neurons were costimulated in vitro in a whole-cell patch-clamp configuration with subthreshold-level short-duration (1-5 ms) electrical and visible light pulses (1-5 ms). The visible light pulses were aimed at a gold-nanoparticle-coated nanoelectrode placed alongside the neuron, within 2 μm distance. Membrane action potentials were recorded with a 3-fold higher success rate and 5-fold better poststimulation cell recovery rate than with pure optical stimulation alone. Also, electrical stimulus current input was being reduced by up to 40%. The subthreshold levels of electrical stimuli in conjunction with visible light (532 nm) reliably triggered trains of action potentials. This single-cell hybrid activation was reliable and repeatable, without any damage as observed with pure optical stimulation. This work represents an empirical cellular study of the membrane action potential response produced by the cultured primary sensory trigeminal neurons when costimulated with plasmonic and electrical (hybrid) stimulation. Our hybrid neurostimulation method can be used toward development of high-acuity neural modulation prosthetic devices, tunable for individual needs, which would qualify as a preferred alternative over traditional electrical stimulation technologies.